The specific gravity of a test sample, such as urine or serum, is a measure of the relative proportions of solid material dissolved in the test sample to the total volume of the test sample. In general, the specific gravity of a test sample is a measure of the relative degree of concentration or the relative degree of dilution of the test sample. With regard to urine samples, the assay for specific gravity, either quantitative or semiquantitative, helps interpret the results of the other assays performed in a routine urinalysis.
Clinically, under appropriate and standardized conditions of fluid restriction or increased fluid intake, the specific gravity of a urine sample measures the concentrating and diluting abilities of the kidneys of an individual. The specific gravity of urine ranges from about 1.005 to about 1.030, and usually is in the range from about 1.010 to about 1.025. A specific gravity of about 1.025 or above in a random first morning urine specimen indicates a normal concentrating ability of the kidneys.
Either an abnormally low or an abnormally high urine specific gravity is clinically significant. Therefore, accurate and reliable specific gravity assays of urine and other aqueous test samples must be available for both laboratory and home use. The assays must provide an accurate measurement of abnormally low and abnormally high specific gravities, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained.
For example, diabetes insipidus, a disease caused by the absence of, or impairment to, the normal functioning of the antidiuretic hormone (ADH), is the most severe example of impaired kidney concentrating ability. This disease is characterized by excreting large urine volumes of low specific gravity. The urine specific gravity of individuals suffering diabetes insipidus usually ranges between 1.001 and 1.003. Low urine specific gravity also occurs in persons suffering from glomerulonephritis, pyelonephritis, and various other renal anomalies. In these cases, the kidney has lost its ability to concentrate the urine because of tubular damage.
An abnormally high urine specific gravity also is indicative of a diseased state. For example, the urine specific gravity is abnormally high in an individual suffering from diabetes mellitus, adrenal insufficiency, hepatic disease or congestive cardiac failure. Urine specific gravity likewise is elevated when an individual has lost an excessive amount of water, such as with sweating, fever, vomiting, and diarrhea. In addition, abnormally high amounts of nonionic urinary constituents, like glucose and protein, increase the urine specific gravity to 1.050 or greater in some individuals suffering from diabetes mellitus or nephrosis. Urine with a fixed low specific gravity of approximately 1.010 that varies little from specimen to specimen is known as isothenuric. This condition is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.
In order to determine if an individual has either an abnormally high or an abnormally low urine specific gravity, and in order to help monitor the course of a medical treatment to determine its effectiveness, simple, accurate and inexpensive specific gravity assays have been developed. In general, the specific gravity of a test sample is a measurement that relates to the density of the test sample. The specific gravity is a value derived from the ratio of the weight of a given volume of a test sample, such as urine, to the weight of the same volume of water under standardized conditions (Eq. 1). ##EQU1## Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts, and organic compounds in water, the specific gravity of urine is greater than 1.000. The relative difference reflects the degree of concentration of the urine specimen and is a measure of the total solids in urine.
Several methods are available to determine the specific gravity of urine. The most widely used method, and possibly the least accurate, employs a urinometer. The urinometer is a weighted, bulb-shaped instrument having a cylindrical stem containing a scale calibrated in specific gravity readings. The urinometer is floated in a cylinder containing the urine sample, and the specific gravity of the urine is determined by the depth the urinometer sinks in the urine sample. The specific gravity value is read directly from the urinometer scale at the junction of the urine with the air. The urinometer method is cumbersome and suffers from the disadvantages of: a) requiring large volumes of urine test sample, b) a difficult and inaccurate reading of the urinometer scale, and c) unreliable assays because the urinometer is not regularly recalibrated.
Refractometry provides an indirect method of measuring the specific gravity of urine. The refractive index of urine is directly related to the number of dissolved particles in urine and, therefore, is directly related to the specific gravity of urine. Consequently, measurement of the refractive index of urine can be correlated to the specific gravity of urine. The refractometer method of determining urine specific gravity is desirable because specific gravity measurements are performed on as little as one drop of urine. However, the refractometer has the disadvantages of requiring daily calibration and not being amenable to home assays.
The falling drop method is another method of assaying for specific gravity which, like the urinometer, directly measures urine specific gravity. In this method, a drop of urine is introduced into each of a series of columns filled with solvent mixtures of increasing and known specific gravity. When the drop of urine comes to rest after its initial momentum has dissipated, and then neither rises nor falls, the specific gravity of the urine is determined to be identical to the specific gravity of the solvent mixture of that particular column. The falling drop method, however, is not widely used in routine urinalysis because of the lengthy time requirements in setting up such a assay and the inability of an individual to perform the assay at home.
The falling drop method described above also can be performed instrumentally. The instrument-based assay uses a specially designed column filled with a silicone oil having a controlled specific gravity and viscosity. The column is designed to measure the time required for a precisely measured drop of test sample to fall a distance defined by two optical gates (lamp-phototransistor pairs) mounted one above the other in a temperature-controlled column filled with a water-immiscible silicone oil of a slightly lower density than the test sample. The falling time is measured electronically and computed into specific gravity units. This specific gravity method is very precise, but the cost of the assay instrument and the degree of skill required to operate the instrument makes home testing for urine specific gravity impractical.
Not one of the above-described specific gravity assay methods is suited to performing specific gravity assays outside a medical office or laboratory. Consequently, reagent impregnated test strips were developed to enable an individual to perform specific gravity assays at home. In general, the test strip assay developed for specific gravity determinations is an indirect assay method, wherein the test strip changes color in response to the ionic strength of the urine sample. The ionic strength of a test sample is a measure of the type and amount of ions present in a test sample. The specific gravity of a test sample is proportional to test sample ionic strength. Therefore, by assaying for the ionic strength of a test sample, the specific gravity is determined indirectly by correlating the ionic strength of the test sample to the specific gravity of the test sample.
The present day specific gravity test strips are pH dependent, and comprise a carrier matrix impregnated with a reagent composition including a polyelectrolyte, such as a partially neutralized poly(methyl vinyl ether/maleic acid); a chromogenic indicator, such as bromothymol blue; and suitable buffering agents. The reagent composition is sensitive to the number of ions, or electrolytes, in the test sample, such that the polyelectrolyte of the reagent composition undergoes an ion exchange, and releases hydrogen ions into the test sample solution in exchange for cations present in the test sample in an amount relative to the ionic strength of the urine sample.
Therefore, as the concentration of electrolytes in urine increases (high specific gravity), more cations are available to exchange with hydrogen ions present on the polyelectrolyte of the reagent composition. The overall result is a release of hydrogen ions into the urine sample, and a resulting pH decrease of the urine sample that causes a color transition of the bromothymol blue chromogenic indicator from blue-green to green to yellow-green in response to increased specific gravity. The resulting color transition, indicating a pH change caused by increasing ionic strength, i.e., increasing specific gravity, is empirically related to the specific gravity of the urine sample.
For test strips utilizing the partially neutralized poly(methyl vinyl ether/maleic acid) polyelectrolyte and bromothymol blue indicator, assays for specific gravity are performed on aqueous test samples having a specific gravity of about 1.000 to about 1.030. A reading of 1.000, or a blue-green color, indicates that the urine has a very low specific gravity, as demonstrated by the lack of a color transition of the chromogenic indicator dye. A specific gravity reading of about 1.005 to about 1.030 is signified by color transitions, from blue-green through green to yellow-green, that serve as reliable indicators of increasing specific gravity.
In accordance with the present day reagent strip method, an individual can readily determine, visually, the specific gravity of a urine sample in the range of about 1.000 to about 1.030. However, the presently available commercial test strips use a pH indicator and are pH dependent. Accordingly, the assay is partially affected by the pH of the urine sample. Therefore, it is desirable to provide a method of determining urine specific gravity that is essentially independent of urine sample pH, such that an accurate specific gravity assay can be interpreted in conjunction with assays for other urine analytes to provide a reliable diagnosis and to allow initiation of a correct medical treatment.
It would be extremely advantageous to have a simple and trustworthy method of assaying for urine specific gravity that allows visual differentiation of specific gravity values from about 1.000 to about 1.035. By providing a semiquantitative method of determining urine specific gravity in an easy to use form, such as a dip-and-read test strip, the urine assay can be performed by laboratory personnel to afford immediate test results. The semiquantitative specific gravity assay results can be interpreted in conjunction with assays for other urine constituents, such that a diagnosis can be made without having to wait for assay results and a medical treatment can be commenced immediately. Furthermore, the test strip method can be performed by an individual at home to semiquantitatively determine the specific gravity of the urine and therefore to help monitor the success of the medical treatment the individual is undergoing.
As will be described more fully hereinafter, the method of the present invention is essentially independent of test sample pH and allows the fast, semiquantitative assay for specific gravity of urine and other aqueous test samples by utilizing a reagent composition comprising a complexing agent, like a polyelectrolyte, an ion exchange material, a chelating agent, or a mixture thereof; a polyvalent metal ion having a valence of at least two; and an indicator capable of interacting with the polyvalent metal ion to provide a color transition. The reagent composition provides sufficient sensitivity and sufficient visual color differentiation between urine samples to yield semiquantitative specific gravity assays. In addition, urine specific gravities of about 1.000 to about 1.035 can be determined quickly.
Any method of assaying for the specific gravity of urine or other aqueous test samples must yield trustworthy and reproducible results by utilizing a reagent composition that undergoes a color transition in response to the specific gravity of the test sample, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with another test sample component, like protein or glucose. Additionally, the method and composition utilized in the specific gravity assay should not adversely affect or interfere with other test reagent pads that are present on multiple test pad strips.
In accordance with the present invention, the reagent composition can be incorporated into the carrier matrix to provide sufficient sensitivity and color differentiation to assay for cation concentration, and therefore for specific gravity between about 1.000 to about 1.035. In addition, although dry phase test strips have been used to assay for specific gravity, no dry phase test strip has incorporated a complexing agent, a polyvalent metal ion, and an indicator capable of interacting with the polyvalent metal ion to provide a color transition in a semiquantitative assay for specific gravity of a test sample.
Prior patents disclose the polyelectrolyte. dye ion exchange chemistry essentially utilized in the specific gravity assay of urine. For example, Falb et al. U.S. Pat. No. 4,318,709 and Stiso et al. U.S. Pat. No. 4,376,827 disclose a polyelectrolyte-dye technique used to assay for urine specific gravity. Each patent teaches utilizing polyelectrolyte-dye chemistry to determine the specific gravity of urine by monitoring the color transition of the dye.
The Falb et al. and Stiso et al. patents each disclose a composition and a method wherein the cations present in the test sample induce an ion exchange with the polyelectrolyte, thereby introducing hydrogen ions into the test solution. The change in hydrogen ion concentration, i.e., pH, is detected by a pH indicator. Accordingly, the previously disclosed methods are sensitive to the pH of the aqueous solution.
The composition and method of the present invention differ from the above disclosures in that a polyvalent metal ion having a valence of at least two first complexes with a complexing agent, like a polyelectrolyte, to form a polyvalent metal ion complex. Then, as a result of contact between the present reagent composition and a test sample including a sufficient concentration of cations, the cations compete with the polyvalent metal ion for the complexing agent and displace a number of the polyvalent metal ions from the complexing agent. The number of polyvalent metal ions displaced from the complexing agent is directly proportional to the concentration of cations in the test sample.
After being displaced from the complexing agent, the polyvalent metal ions are available to interact with the indicator and form a polyvalent metal ion-indicator complex. The polyvalent metal ion-indicator complex is different in color from the reagent composition and test sample, and therefore provides a detectable and measurable color transition. The color transition can be correlated to the specific gravity of the test sample because the color transition is directly proportional to the amount of polyvalent metal ion released from the complexing agent, which in turn is directly proportional to the cation concentration of the test sample. The cation concentration of the test sample is directly proportional to test sample specific gravity. Accordingly, and in contrast to the Falb et al. and Stiso et al. disclosures, the present method is essentially independent of test sample pH because the color transition results from a pH-independent displacement of polyvalent metal ions, such as ferrous ions or cobaltous ions, from a complexing agent, such as a polyelectrolyte, an ion exchange material or a chelating agent.
The present invention provides a composition and method for semiquantitatively determining the specific gravity of urine and other aqueous test samples by utilizing a reagent composition including an indicator capable of forming a complex with the polyvalent metal ion and providing a color transition. European Patent Application 0 349 934 discloses a test strip and method of determining specific gravity of a sample utilizing a composition including a buffer, a complex former and a pH indicator dye. The complex former can be a crown ether, a cryptand, a podand or a multifunctional ligand. The method disclosed in the European Application is pH dependent, and utilizes a standard pH indicator dye, such as bromothymol blue or thymol blue. European Patent Application 0 349 934 does not teach or suggest a combination of a complexing agent, a polyvalent metal ion and an indicator, as utilized in the present invention, to provide an essentially pH independent assay for specific gravity.
In contrast to the above-described patents, and in contrast to the presently available commercial test strips, the method of the present invention provides a semiquantitative measurement of test sample specific gravity by utilizing a reagent composition including a complexing agent, like a polyelectrolyte, an ion exchange material, a chelating agent, or a mixture thereof; a polyvalent metal ion having a valence of at least two; and an indicator capable of interacting with the polyvalent metal ion to provide a color transition, wherein the method is essentially independent of test sample pH. The present reagent composition undergoes a sufficient color transition upon contact with a test sample to provide a semiquantitative specific gravity assay for liquids having a specific gravity of about 1.000 to about 1.035. Hence, new and unexpected results are achieved in the wet phase and the dry phase reagent strip assay of urine and other aqueous test samples for specific gravity.